Microarrays as a functional approach to the transcriptome

Knowing a cell’s transcriptome is a fundamental requisite in order to analyze its response to the environment. Microarrays have supposed a revolution on this field as they are able to yield an overview of gene expression at any environmental condition on a genome-wide scale. This technique consists in the hybridisation of a nucleic acid sample, previously marked, with a probe (which might be made up of cDNA, oligonucleotides or PCR products) anchored to a solid surface (made of glass, plastic, silicon...) giving as a result a dot grid which reveals, after image analysis, which genes are being expressed. Nevertheless, this only can be achieved if information on the species genome has been generated. Different kinds of expression microarrays exist attending to the probe’s nature and the method used in its synthesis. In this poster two of these will be treated: Spotted Microarrays, for which the probe is synthesised prior to its fixation to the array and allow the analysis of two targets simultaneously. They can be easily customized, but lack high reproducibility and sensitivity. Oligonucleotide Microarrays, which are characterized by the direct printing of the probe on the array. In this case the probes consist on, invariably, oligonucleotides that are complementary to a small fraction of the gene it is representing at the microarray. Their application is somewhat restricted. This fact, however, makes them more reproducible. Currently, the approach towards the transcriptome studies from the Next Generation Sequencing technologies offers a large volume of information in a short amount of time needing less previous information on the target organism than that needed by microarrays, but their expensive price limits their use. The versatility of the latter, together with their reduced costs in comparison to other techniques, makes them an interesting resource in applications that may need less complexity

miRNA/phasiRNA mediated regulation of plant defense response against P. syringae

Gene silencing is a mechanism of regulation of gene expression where the small RNAs (sRNAs) are key components for giving specificity to the system. In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs). DCL proteins acting on large RNA precursors produce the mature forms of sRNAs (20-24nt) that can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have validated this regulation, and have also established that the corresponding pri-miRNA is down-regulated upon PAMPs or bacteria perception. Using GUS reporters, we have characterized the expression pattern of both pri-miRNA and its best target R genes. We demonstrate that plants with altered levels of miRNA* (knockdown or overexpression lines) exhibit altered PTI-associated phenotypes, supporting a role for this miRNA* in the defence response against this bacterial pathogen. Finally, we identify phasiRNAs that arise from the transcript of one of the R target genes in a miRNA*-RDR6-DCL4-dependent manner.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Caracterización de la hipotética proteína de defensa codificada por el gen At5G38850 en Arabidopsis thaliana

La defensa PTI (Pattern triggered immunity) en plantas evita la infección por parte de un gran número de patógenos. P. syringae suprime la PTI mediante proteínas (efectores) introducidas en el interior de la célula vegetal por un sistema de secreción tipo III. La ETI (Effector triggered immunity), detecta la actividad de los efectores, mediante receptores intracelulares denominados proteínas “R”. En plantas, los pequeños RNAs participan en regulación de la expresión mediante silenciamiento génico. Existen dos tipos de pequeños RNAs en silenciamiento génico: pequeños RNAs interferentes (siRNAs) y microRNAs (miRNAs). Los miRNAs son RNAs no codificantes de cadena sencilla que reducir la expresión de transcritos mediante la unión al complejo RISC (RNA-induced silencing complex) por un mecanismo dependiente de secuencia. Este mecanismo regula las respuestas de defensa frente a muchos patógenos. Nosotros trabajamos con miR825* de A. thaliana que presenta como dianas genes “R” aún por caracterizar. MiR825* regula la expresión de estos genes R y desencadena la generación de pequeños RNAs en fase (phasiRNAs) a partir de una de estas dianas. La activación de PTI determina una bajada en los niveles de miRNA825* , activándo así la expresión de estos genes R. Plantas con niveles alterados de miRNA825* muestran una PTI alterada. Hemos seleccionado una de las dianas reguladas por este miRNA, el gen AT5G38850, y hemos mostrado mediante fusiones a GFP y. microscopia confocal, que se localiza tanto en el núcleo como en el citoplasma. También hemos caracterizado el patrón de expresión del gen y del miRNA, demostrando que este último regula los niveles del primero en diferentes tejidos. La expresión inducible de la proteína en los fondos Col-0 (silvestre) y DCL234 (mutante afectado en la biogénesis de siRNAs) lleva a su acumulación solo en el fondo mutante , sugierendo un mecanismo de autorregulación mediado por phasiRNAs.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Molecular characterization of MicroRNA-silenced TNL-1 (MIST1) and its role in plant defense

miRNAs are sequence-dependent negative regulators of gene expression involved in many relevant plant processes, including immunity. Activation of defence genes can negatively impact plant fitness, and thus needs to be fine-tuned. miRNA-mediated regulation of gene expression is mediated by the activity of DCL proteins that induce cleavage of target transcripts. We have described miR825-5p as involved in the regulation of immunity. This miRNA specifically targets R genes of the TNL family. We have characterized the regulatory system formed by miRNA825-5p and its main target MIST1. This miRNA only triggers the RDR6-DCL4-dependent production of phasiRNAs from MIST1. MIST1 is the NLR gene from which the most phasiRNAs are produced in Arabidopsis. We reported that pri-miR825 is down-regulated after PAMP-perception and demonstrated that plants with altered levels of miR825-5p exhibit altered PTI-associated phenotypes. In addition, MIST1 has been described to be regulated by other mechanisms like nonsense mediated decay or polyubiquitination. We have characterized the expression pattern of both MIR825 and MIST1 and are currently studying the putative molecular role of MIST1 in defence apart from its demonstrated role as a miRNA825-5p-linked regulatory hub for TNLs regulation in Arabidopsis thaliana.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

The When and Where: How development modulates immunity through a miRNA-NLR-phasiRNA network

Plants possess a battery of dedicated intracellular immune receptors known as nucleotide-binding leucine-rich repeat proteins (NLRs) involved in recognition of pathogen-derived effectors and activation of effector-triggered immunity (ETI). In a recent work, we report a two-tiered regulatory network in Arabidopsis mediated by a microRNA (miR825-5p) and a wave of secondary NLR-derived phased small RNAs (phasi-NLRs) involved in silencing dozens of NLR genes encoding Toll/interleukin-1 NLRs (TNLs). In our model, targeting of MIST1 (microRNA-silenced TNL1) transcripts by miR825-5p, triggers the generation of phasi-NLRs that subsequently silence a wide network of TNL-encoding genes and reinforce the silencing of MIST1. Current knowledge regarding how these networks are modulated contribute to immunity during different developmental stages is scarce. Through generation of GUS-based A. thaliana reporter lines we have characterized the expression pattern of both MIR825 and MIST1 genes and the domain activity of miR825-5p. A comprehensive analysis of RNA and small RNA-seq datasets, unravelled a hitherto unknown connection between miR825-5p/phasi-NLR mediated regulation of NLRs and plant development. Further analysis supports the notion that the action of this regulatory mechanism is restricted to control NLR expression in leaves. We are working to understand how the changes on miRNA/phasi-NLR levels during developmental stages modulate the immune response and the biological impact of this developmental-dependent regulation on plant health. Our results point to a developmentally regulated sRNA-based control of NLR expression in Arabidopsis and shed light on how levels of these regulatory molecules (miRNAs and phasi-NLRs) are modulated during development or in an organ specific manner to fine-tune NLR expression.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

R gene regulation mediated by miRNA/phasiRNA during plant defense response against P. syringae

In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs), differing these in their biogenesis and mode of action, but sharing similar sizes (20-24 nt). In plants, their mature forms are products of the activity of DCL proteins and can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. By bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception. We demonstrate that plants with altered levels of this miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. We have characterized the expression pattern of both primiRNA and its best target R genes. Finally, we identify phasiRNAs that arise from the transcript of this R gen in a miRNA*-RDR6-DCL4-dependent mannerUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Regulación de genes de resistencia de la familia TIR-NBS-LRR mediada por miRNA/phasiRNA durante la interacción con P. syringae

Durante un estrés biótico, las plantas modulan la expresión de una batería de genes involucrados en la respuesta de defensa, proceso donde recientemente se ha determinado el papel esencial que desempeña el silenciamiento génico. El silenciamiento génico es un mecanismo de regulación de la expresión génica, donde destacan como principales moléculas efectoras los pequeños RNAs (sRNAs). En plantas, estos sRNAs, son clasificados en pequeños RNAs interferentes (siRNAs) o microRNAs (miRNAs), presentando tamaños similares (20-24 nt) pero difiriendo en su biogénesis y modo de acción. Los miRNAs son pequeños RNAs de cadena sencilla que actúan regulando negativamente la expresión de genes, mediante su unión al complejo RISC (Rna Induced Silencing Complex) y en una forma dependiente de secuencia. En nuestro laboratorio, mediante el análisis de datos transcriptómicos, y el uso de herramientas bioinformáticas, identificamos un miRNA* de 22 nt como potencial regulador de la expresión de genes de resistencia (“R”) del tipo TIR-NBS-LRR. Posteriormente hemos validado dicha regulación y caracterizado los patrones de expresión tanto del Pri-miRNA como de un gen “R” regulado por este, en diferentes tejidos y estadios del desarrollo, así como durante la interacción con P. syringae. Por otro lado, hemos generado plantas transgénicas que presentan niveles alterados del miRNA* (incremento y reducción) y hemos observado que muestran fenotipos alterados de PTI y una mayor/menor colonización de P. syringae. Finalmente hemos identificado la producción de sRNAs (phasiRNAs) a partir del gen de resistencia, en una forma dependiente de miRNA*-RDR6-DCL4, pudiendo estos sRNAs secundarios regular otros transcritos de la misma familia de genes de resistencia.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Variation in Fish Abundance, Diversity and Assemblage Structure in Seagrass Meadows across the Atlanto-Mediterranean Province

Seagrasses worldwide provide key habitats for fish assemblages. Biogeographical disparities in ocean climate conditions and seasonal regimes are well-known drivers of the spatial and temporal variation in seagrass structure, with potential effects on associated fish assemblages. Whether taxonomically disparate fish assemblages support a similar range of ecological functions remains poorly tested in seagrass ecosystems. In this study, we examined variation in the abundance, diversity (from a taxonomic and functional perspective), and assemblage structure of fish community inhabiting nine meadows of the seagrass Cymodocea nodosa across three regions in the Mediterranean (Mallorca and Alicante) and the adjacent Atlantic (Gran Canaria), and identified which attributes typifying the structure of meadows, and large-scale variability in ocean climate, contributed most to explaining such ecological variation. Despite a similar total number of species between Mallorca and Gran Canaria, the latter region had more taxonomically and functionally diverse fish assemblages relative to the western Mediterranean regions, which translated into differences in multivariate assemblage structure. While variation in the abundance of the most conspicuous fish species was largely explained by variation in seagrass structural descriptors, most variation in diversity was accounted for by a descriptor of ocean climate (mean seasonal SST), operating at regional scales. Variation in fish assemblage structure was, to a lesser extent, also explained by local variability in seagrass structure. Beyond climatic drivers, our results suggest that lower temporal variability in the canopy structure of C. nodosa meadows in Gran Canaria provides a more consistent source of food and protection for associated fish assemblages, which likely enhances the more abundant and diverse fish assemblages thereEn prens

Effect of an Intensive Weight-Loss Lifestyle Intervention on Kidney Function: A Randomized Controlled Trial

Introduction: Large randomized trials testing the effect of a multifactorial weight-loss lifestyle intervention including Mediterranean diet (MedDiet) on renal function are lacking. Here, we evaluated the 1-year efficacy of an intensive weight-loss intervention with an energy-reduced MedDiet (erMedDiet) plus increased physical activity (PA) on renal function. Methods: Randomized controlled "PREvención con DIeta MEDiterránea-Plus"(PREDIMED-Plus) trial is conducted in 23 Spanish centers comprising 208 primary care clinics. Overweight/obese (n = 6,719) adults aged 55-75 years with metabolic syndrome were randomly assigned (1:1) to an intensive weight-loss lifestyle intervention with an erMedDiet, PA promotion, and behavioral support (intervention) or usual-care advice to adhere to an energy-unrestricted MedDiet (control) between September 2013 and December 2016. The primary outcome was 1-year change in estimated glomerular filtration rate (EGFR). Secondary outcomes were changes in urine albumin-to-creatinine ratio (UACR), incidence of moderately/severely impaired EGFR (<60 mL/min/1.73 m2) and micro-to macroalbuminuria (UACR ≥30 mg/g), and reversion of moderately (45 to <60 mL/min/1.73 m2) to mildly impaired GFR (60 to <90 mL/min/1.73 m2) or micro-to macroalbuminuria. Results: After 1 year, EGFR declined by 0.66 and 1.25 mL/min/1.73 m2 in the intervention and control groups, respectively (mean difference, 0.58 mL/min/1.73 m2; 95% CI: 0.15-1.02). There were no between-group differences in mean UACR or micro-to macroalbuminuria changes. Moderately/severely impaired EGFR incidence and reversion of moderately to mildly impaired GFR were 40% lower (HR 0.60; 0.44-0.82) and 92% higher (HR 1.92; 1.35-2.73), respectively, in the intervention group. Conclusions: The PREDIMED-Plus lifestyle intervention approach may preserve renal function and delay CKD progression in overweight/obese adults.This work was supported by the official Spanish Institutions for funding scientific biomedical research, CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN) and Instituto de Salud Carlos III (ISCIII), through the Fondo de Investigación para la Salud (FIS), which is cofunded by the European Regional Development Fund (5 coordinated FIS projects leaded by J.S.-S and J.V., including the following projects: PI13/00673, PI13/00492, PI13/00272, PI13/01123, PI13/00462, PI13/00233, PI13/02184, PI13/00728, PI13/01090, PI13/01056, PI14/01722, PI14/00636, PI14/00618, PI14/00696, PI14/01206, PI14/01919, PI14/00853, PI14/01374, PI14/00972, PI14/00728, PI14/01471, PI16/00473, PI16/00662, PI16/01873, PI16/01094, PI16/00501, PI16/00533, PI16/00381, PI16/00366, PI16/01522, PI16/01120, PI17/00764, PI17/01183, PI17/00855, PI17/01347, PI17/00525, PI17/01827, PI17/00532, PI17/00215, PI17/01441, PI17/00508, PI17/01732, PI17/00926; PI19/00957, PI19/00386, PI19/00309, PI19/01032, PI19/00576, PI19/00017, PI19/01226, PI19/00781, PI19/01560, and PI19/01332); the Especial Action Project entitled Implementación y evaluación de una intervención intensiva sobre la actividad física Cohorte PREDIMED-Plus grant to J.S.-S.; the European Research Council (Advanced Research Grant 2014–2019; agreement #340918) granted to M.Á.M.-G.; the Recercaixa (No. 2013ACUP00194) grant to J.S.-S.; grants from the Consejería de Salud de la Junta de Andalucía (PI0458/2013, PS0358/2016, and PI0137/2018); the PROMETEO/2017/017 grant from the Generalitat Valenciana; the SEMERGEN grant; funds from the European Regional Development Fund (CB06/03); International Nut & Dried Fruit Council – FESNAD (Long-term effects of an energyrestricted Mediterranean diet on mortality and cardiovascular disease 2014–2015, No. 201302) (PI: M.Á.M.-G.); the AstraZeneca Young Investigators Award in Category of Obesity and T2D 2017 (PI: D.R.); grant of support to research groups No. 35/2011 (Balearic Islands Gov.; FEDER funds) (J.A.T. and C.B.); the JR17/00022 (ISCIII) grant to O.C.; the Boosting young talent call grant program for the development of IISPV research projects 2019–2021 (Ref.: 2019/IISPV/03 grant to A.D.-L.); the Societat Catalana d’Endocrinologia i Nutrició (SCEN) Clinical-Research Grant 2019 (IPs: J.S.-S. and A.D.-L.). Collaborative Nutrition and/or Obesity Project for Young Researchers 2019 supported by CIBEROBN entitled Lifestyle Interventions and Chronic Kidney Disease: Inflammation, Oxidative Stress and Metabolomic Profile (LIKIDI study) grant to A.D.-L

Detection of kinase domain mutations in BCR::ABL1 leukemia by ultra-deep sequencing of genomic DNA

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E−4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E−4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.Tis project was funded in part by CRIS CANCER FOUNDATION